Acyl carrier protein-I/protein-A gene fusion, products and methods

Abstract

A gene encoding spinach acyl carrier protein-I-Gly76 (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. The gene fusion can be expressed in E. coli thereby resulting in the production of an appropriately sized protein possessing ACP activity in acyl ACP synthetase reactions, plant MCT reactions, and plant fatty acid synthesis. This gene fusion, under the control of the lambda PR promoter, expresses high levels of a 42 kDa fusion protein in E. coli. This protein functions as a affinity moiety in purification methods for the isolation of enzymes involved in fatty acid synthetase, acyl ACP synthetase and malonyl-CoA:ACP tranacylase reactions.

We claim: 
     
       1. A recombinant DNA which encodes a plant acyl carrier protein and the F c  -binding portion of staphylococcal protein A, wherein the recombinant DNA comprises a first sequence encoding the acyl carrier protein gene downstream and in frame with a second DNA sequence encoding the F c  -binding portion of staphylococcal protein A. 
     
     
       2. A recombinant DNA of claim 1, wherein the plant acyl carrier protein is a spinach acyl carrier protein. 
     
     
       3. A recombinant DNA of claim 2, wherein the spinach acyl carrier protein is spinach acyl carrier protein-I-Gly 76 . 
     
     
       4. The recombinant DNA of claim 3 wherein said protein is further characterized in that the level of fatty acid synthetase activity is approximately proportional to the level of acyl carrier protein throughout the range of from 0 to about 20 μM. 
     
     
       5. A recombinant DNA construct comprising a first DNA sequence which encodes a plant acyl carrier protein and a second DNA sequence which encodes the F c  -binding portion of staphylococcal protein A, wherein the first DNA encoding the acyl carrier protein gene is downstream and in frame with the DNA encoding the F c  -binding portion of staphylococcal protein A. 
     
     
       6. The construct of claim 5, wherein said construct is a plasmid. 
     
     
       7. The construct of claim 5, wherein said construct is a cloning vector. 
     
     
       8. The construct of claim 5, wherein said construct is an expression vector. 
     
     
       9. The recombinant DNA construct of claim 5, wherein the plant acyl carrier protein is a spinach acyl carrier protein. 
     
     
       10. The construct of claim 9, wherein said construct is a plasmid. 
     
     
       11. The construct of claim 9, wherein said construct is a cloning vector. 
     
     
       12. The construct of claim 9, wherein said construct is an expression vector. 
     
     
       13. The recombinant DNA construct of claim 9, wherein the plant acyl carrier protein is spinach acyl carrier protein-I-Gly 76 . 
     
     
       14. The construct of claim 13, wherein said construct is a plasmid. 
     
     
       15. The construct of claim 13, wherein said construct is a cloning vector. 
     
     
       16. The construct of claim 13, wherein said construct is an expression vector. 
     
     
       17. The recombinant DNA construct of claim 13 wherein said protein is further characterized in that the level of fatty acid synthetase activity is approximately proportional to the level of acyl carrier protein throughout the range of from 0 to about 20 μM. 
     
     
       18. A phase comprising the recombinant DNA construct of claim 16. 
     
     
       19. A method of producing an enzymatically active acyl carrier protein bound to the F c  -binding portion of staphylococcal A protein comprising: a. transforming cells for expressing said protein with an expression vector carrying a recombinant DNA comprising a first sequence encoding the acyl carrier protein gene downstream and in frame with a second DNA sequence encoding the F c  -binding portion of staphylococcal protein A; and   b. culturing said cells under conditions wherein said proteins are expressed.   
     
     
       20. The method of claim 19 wherein the protein is spinach acyl carrier protein-I-Gly 76  characterized in that the level of fatty acid synthetase activity is approximately proportional to the level of acyl carrier protein throughout the range of from 0 to about 20 μM.